Journal: Nucleic Acids Research
Article Title: A high-throughput DNA methylation analysis of a single cell
Figure Lengend Snippet: Principle of the RSMA test system for the analysis of DNA methylation patterns in single cells. ( A ) Single cells from various sources are placed on high-throughput multi-well PCR slides (one cell per well), either by micromanipulation, laser capture microdissection or flow cytometry cell sorting. The protocol was tested with AmpliGrid microreaction slides, which are composed of a standard microscope slide sized glass surface with chemically structured, DNA free reaction centers. The reaction volume ranges from ∼500 nl up to 1.65 μl, small enough to sufficiently concentrate the reaction volume to a point where enzymatic steps proceed with high kinetics, but large enough to sufficiently dilute the cellular components (∼1 pl) of each cell. It is possible to perform 48 methylation-profiling reactions on one 75 mm × 25 mm slide. The structure of the slides allows carrying out methylation-sensitive PCRs from single cells that can be lysed directly on the reaction centers, thereby generating methylation profiles starting with DNA amounts as low as 6 pg. The hydrophilic and hydrophobic regions on the slide surface ensure that even after inaccurate pipetting the droplets containing single cells will find their way to the correct position. An important feature of this technology is the parallel analysis of multiple cells, a desirable feature that makes it possible to obtain statistically relevant methylation data on single cells in a reasonable time frame. ( B ) DNA is fragmented by a restriction enzyme (here NlaIV), that cleaves outside of the target region. Primer mixes contain two forward primers (F1 + F2) and one reverse primer (R). The analyzed CpG dinucleotides (here HpaII and Hin6I sites) are located between the two forward primers. If the restriction site is methylated, the restriction enzyme cannot cleave and both PCR products are synthesized, whereas for unmethylated restriction sites only the short PCR product (internal PCR positive control) is produced.
Article Snippet: Living single cells (propidium iodide negative) in 1 × PBS were deposited on the reaction sites of AmpliGrid slides (Beckman Coulter Biomedical GmbH, Advalytix Products, Munich/Germany, BCB) according to their physical properties (size, granularity) with a fluorescence-activated cell sorter (FACS), fitted with an AmpliGrid slide holder (BCG).
Techniques: DNA Methylation Assay, High Throughput Screening Assay, Polymerase Chain Reaction, Micromanipulation, Laser Capture Microdissection, Flow Cytometry, Cytometry, FACS, Microscopy, Methylation, Synthesized, Positive Control, Produced